Evaluation of the tetrodotoxin uptake ability of pufferfish Takifugu rubripes tissues according to age using an in vitro tissue slice incubation method

The tetrodotoxin (TTX) uptake ability of pufferfish Takifugu rubripes tissues and its growth-associated changes were investigated using an in vitro tissue slice incubation method. Tissue slices prepared from the liver, skin, and intestine of a non-toxic cultured adult T. rubripes (20 months old) and incubated with incubation buffer containing 25 μg/mL TTX for 1?48 h showed a time-dependent increase in the TTX content in all tissues. The TTX contents of the skin and intestine slices were comparable to or slightly higher than that of the liver slices, with a similar transition pattern, suggesting similar TTX uptake ability among the skin, intestine, and liver. The TTX uptake ability of the liver and intestine did not differ significantly between young (8 months old) and adult (20 months old) fish, but the skin slices of young fish took up approximately twice as much TTX as that of adult fish, suggesting that the TTX uptake ability of the skin is involved in the growth-dependent changes in the toxin distribution inside the body in T. rubripes. To estimate the TTX uptake pathway in each tissue, an immunohistochemical technique was used to observe temporal changes in the intra-tissue microdistribution of TTX during incubation. The findings suggested that TTX is transferred and accumulates from pancreatic exocrine cells to hepatic parenchymal cells in the liver, from connective tissues to basal cells in the skin, and from villi epithelial cells via the lamina propria to the muscle layer in the intestine

Transfer profile of intramuscularly administered tetrodotoxin to artificial hybrid specimens of pufferfish, Takifugu rubripes and Takifugu niphobles.

Tetrodotoxin (TTX) was intramuscularly administered to artificially hybridized specimens of the pufferfish Takifugu rubripes and Takifugu niphobles to investigate toxin accumulation in hybrids, and TTX transfer/accumulation profiles in the pufferfish body. In the test fish administered 146 MU TTX in physiologic saline, TTX rapidly transferred from the muscle via the blood to other organs. Toxin transfer to the ovary rapidly increased to 53.5 MU/g tissue at the end of the 72-h test period. The TTX content in the liver and skin was, at most, around 4-6 MU/g tissue, and in the testis it was less than 0.01 MU/g tissue. On the other hand, based on the total amount of toxin per individual (% of the administered toxin), the skin and the liver contained higher amounts (20-54% and 2-24%, respectively), but the amount in the liver rapidly decreased after 8-12 h, and fell below the level in the ovary after 48 h. These findings suggest that part of the TTX is first taken up in the liver and then transferred/accumulated in the skin in male specimens and in the ovary in female specimens

Larval pufferfish protected by maternal tetrodotoxin

Marine pufferfish contain tetrodotoxin (TTX), an extremely potent neurotoxin. All species of the genus Takifugu accumulate TTX in the liver and ovaries, although the tissue(s) in which it is localized can differ among species. TTX is the major defense strategy the pufferfish appears to use against predators. TTX is also used as a male-attracting pheromone during spawning. Here we demonstrate an additional (and unexpected) use of maternal TTX in the early larval stages of the Takifugu pufferfish. Predation experiments demonstrated that juveniles of all the species of fish used as predators ingested pufferfish larvae, but spat them out promptly. Liquid Chromatography-Tandem Mass Spectrometry (LC-MSMS) analysis revealed that the pufferfish larvae contain a small quantity of TTX, which is not enough to be lethal to the predators. Immunohistochemical analysis with anti-TTX monoclonal antibody revealed that the TTX is primarily localized in the body surface of the larvae as a layer of protection. Our study showed the female parent of the Takifugu pufferfish vertically transfers TTX to the larvae through its accumulation in the ovaries, and subsequent localization on the body surface of the larvae

Co-Occurrence of Tetrodotoxin and Saxitoxins and Their Intra-Body Distribution in the Pufferfish Canthigaster valentini

Abstract: Pufferfish of the family Tetraodontidae possess tetrodotoxin (TTX) and/or saxitoxins (STXs), but the toxin ratio differs, depending on the genus or species. In the present study, to clarify the distribution profile of TTX and STXs in Tetraodontidae, we investigated the composition and intra-body distribution of the toxins in Canthigaster valentini. C. valentini specimens (four male and six female) were collected from Amami-Oshima Island, Kagoshima Prefecture, Japan, and the toxins were extracted from the muscle, liver, intestine, gallbladder, gonads, and skin. Analysis of the extracts for TTX by liquid chromatography tandem mass spectrometry and of STXs by high-performance liquid chromatography with post-column fluorescence derivatization revealed TTX, as well as a large amount of STXs, with neoSTX as the main component and dicarbamoylSTX and STX itself as minor components, in the skin and ovary. The toxins were also detected in the other tissues, but in much lower amounts than in the skin and ovary. The TTX/STX ratio varied greatly, depending on the tissue, but TTX was the major toxin component in the whole body, and STXs accounted for 25% and 13% of the total toxin amount in males and females, respectively. Like the marine pufferfish of the genus Arothron, C. valentini should be considered a pufferfish with considerable amounts of both TTX and STXs present simultaneously

Evaluation of the tetrodotoxin uptake ability of pufferfish Takifugu rubripes tissues according to age using an in vitro tissue slice incubation method

The tetrodotoxin (TTX) uptake ability of pufferfish Takifugu rubripes tissues and its growth-associated changes were investigated using an in vitro tissue slice incubation method. Tissue slices prepared from the liver, skin, and intestine of a non-toxic cultured adult T. rubripes (20 months old) and incubated with incubation buffer containing 25 μg/mL TTX for 1–48 h showed a time-dependent increase in the TTX content in all tissues. The TTX contents of the skin and intestine slices were comparable to or slightly higher than that of the liver slices, with a similar transition pattern, suggesting similar TTX uptake ability among the skin, intestine, and liver. The TTX uptake ability of the liver and intestine did not differ significantly between young (8 months old) and adult (20 months old) fish, but the skin slices of young fish took up approximately twice as much TTX as that of adult fish, suggesting that the TTX uptake ability of the skin is involved in the growth-dependent changes in the toxin distribution inside the body in T. rubripes. To estimate the TTX uptake pathway in each tissue, an immunohistochemical technique was used to observe temporal changes in the intra-tissue microdistribution of TTX during incubation. The findings suggested that TTX is transferred and accumulates from pancreatic exocrine cells to hepatic parenchymal cells in the liver, from connective tissues to basal cells in the skin, and from villi epithelial cells via the lamina propria to the muscle layer in the intestine

Additional file 6: Figure S4. of CASTIN: a system for comprehensive analysis of cancer-stromal interactome

FABP5/Fabp5 expression in RNA-Seq (estimated by CASTIN) and Immunohistochemistry. (a) gene expression levels. (b) Immunohistochemical (IHC) and hematoxylin and eosin (H&E) staining of close sections. Sections of PDAC xenograft cancer derived from each cell line were stained for FABP5/Fabp5 (left) or with H&E (right). The slightly different distribution of tumor and stromal cells between H&E and the corresponding IHC sections was due to the physical distance between the two sections. (PDF 10295 kb